Animal model for perimenopause and menopause and methods of inducing ovarian failure

ABSTRACT

The present invention relates to an animal model for human perimenopause and menopause. Also provided by the present invention are methods of making the animal model and methods of screening using the model. Also provided are methods of inducing ovarian failure in animals such as pets and wildlife.

CONTINUING APPLICATION DATA

This application claims the benefit of the filing date of U.S.provisional application Ser. No. 60/406,671, filed on Aug. 29, 2002, thecontents of which are incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was supported by NIH via grant numbers RO1-ES9246,RO1-ES8979, and RO1-AG021948. The government may have certain rights tothis technology.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to an animal model for human perimenopauseand menopause. Also provided by the present invention are methods ofmaking the animal model and methods of screening and using the model.Also provided are methods of inducing ovarian failure in animals such aspets and wildlife.

2. Description of the Background

The average age of menopause in women in the U.S. is 51 years.Demographic studies on the age of menopause have shown that it hasincreased from about 45 years in 1850, to approximately 51 in 1995. Atthe same time, however, the life expectancy of women has increased from45 in 1850 to approximately 82 in 1998. As a result, because the lifespan in women has increased, almost 30% of a woman's lifetime will bepostmenopausal (1). The consequence of this shift is that manyage-related diseases are increasing in incidence and need to beinvestigated in relevant animal models to understand the effect ofmenopause on disease risk, presentation and progression. Many disorderssuch as Alzheimer's disease have an increased incidence in females of arelatively late onset (approximately 80 years of age). Obviously, in thefuture, the increase in life expectancy will impact the incidence ofmany age-related diseases and require aggressive intervention during thepostmenopausal years.

Many health risks are known to be associated with menopause. There is astrong direct link between menopause and an increase in cardiovasculardisease which is the leading cause of death in women over the age of 50(2). A number of observational studies have, provided evidence thathormone replacement therapy (HRT) reduces the risk of cardiovasculardisease by about one-half (3). However, the HERS study recently reportedthat HRT in post menopausal women did not prevent recurrent myocardialinfarction (4). Recently, the Womens Health Initiative study conductedby the NIH reported a slight increase in cardiovasculardisease-associated conditions in women taking HRT (5).

Furthermore, there is a significant debate over the advantages anddisadvantages of using HRT in postmenopausal women relative to apotential increase in both breast and ovarian cancer risks (5, 6, 7).Clearly, studies utilizing a relevant animal model would contributegreatly to resolving these issues. Interestingly, althoughcontroversial, it has been suggested that there are benefits rather thanrisks associated with the estrogens in birth-control pill usage inpremenopausal women (7). These seemingly disparate effects of estrogentreatment will best be resolved in the laboratory.

Menopause is the cessation of ovarian cyclicity resulting from thedepletion of ovarian follicles by a natural process of atrition, knownas atresia (8). Follicular maturation in the ovary is a dynamic seriesof events in which primordial follicles provide a finite pool from whichpreovulatory follicles are selected for development and ovulation, orare eliminated by atresia. The primordial follicle, the most immaturestage of development, is formed in the ovary during fetal development.Because the oocyte is arrested in meiosis, this pool is non-regeneratingafter birth. In an on-going process, after puberty, folliclescontinually progress from the primordial to ovulatory stages. However,the vast majority do not develop to ovulation, but undergo cell death byatresia. As a result, the pool of primordial follicles gradually becomesdepleted and ultimately, ovarian failure (menopause) ensues (8). As thepool of primordial follicles is depleted, this compromises the numbersof developing pre-ovulatory follicles. Eventually, the reduction inpre-ovulatory follicles significantly alters ovarian steroid hormoneproduction as a woman approaches menopause (perimeriopause), resultingin a sharp decrease in circulating 17β-estradiol and a concomitant risein the gonadotropins follicle stimulating hormone (FSH) and luteinizinghormone (LH), due to loss of negative feedback from the ovary to thepituitary. Thus, following menopause, hormonal cyclicity ceases withinthe ovary and it secretes primarily androgens in a hypergonadotropicenvironment (8). Because 17β-estradiol is assumed to afford protectionin premenopausal women against health risks, such as cardiovasculardisease, skeletal problems, and brain dysfunction, the loss of17β-estradiol in menopause is thought to contribute to mostmenopause-associated disorders.

In researching menopause, a limited amount of mechanistic informationcan be obtained from studies in middle-aged women. Therefore, theelucidation of underlying cellular and molecular mechanisms thataccompany menopause-associated disorders requires the use of appropriateanimal models in controlled experimental conditions. Although nonhumanprimates most closely resemble humans, there are disadvantages in usingthem in the study of menopause. These include limitations on the numberof animals, costly acquisition and housing expenses, and lengthy lifespans, with reproductive senescence occurring late in life (9). Rodentmodels, on the other hand, are inexpensive and reproductive senescencecan be caused and studied within a relatively short time frame. Previousrodent studies have attempted to model menopause by ovariectomy. Whilethis approach mimics the loss of 17β-estradiol seen in menopause, itlacks consideration of the physiological contributions of thepostmenopausal ovary. It is possible that the postmenopausal ovaryimpacts the effects of the changing hormonal and gonadotropin milieu viasecretion of bioregulatory factors. In postmenopausal women who have hadtheir ovaries removed, a 50% reduction in testosterone has beenobserved, indicating that the senescent ovary is secreting androgensthat have the potential to impact postmenopausal health risks (10).However, because there has not heretofore been an adequate animal modelof the postmenopausal ovary, this issue has not been directlyinvestigated. In surgical experiments conducted in mice, aged ovarieswere transplanted into young ovex recipients. Plasma FSH levels weresignificantly increased compared to controls, indicating that the agedovary plays a role in reproductive failure by impacting thehypothalamic-pituitary-ovarian axis, perhaps by as yet-unknownbioregulatory factors as well as loss of negative feedback due to areduction in 17β-estradiol. Collectively, the lack of information aboutthe follicle-depleted ovary supports the need for investigations intoits function in vivo as well as in vitro to understand the effects onage-related diseases associated with menopause.

The occupational chemical VCD, the diepoxide metabolite of4-vinylcyclohexene (VCH), causes selective loss of small preantralfollicles in the ovaries of mice and rats (11-13). Compared to vehiclecontrols, VCD dosing of rats and mice for 12 days caused a significantreduction in the number of primordial and primary follicles (12).However, following 30 days of dosing there was also a significantreduction in the numbers of secondary follicles (11, 13) which wasexplained as a reduction in the pool of primordial follicles from whichsecondary follicles could be recruited at that time to develop intolarge antral follicles that produce 17β-estradiol. The targeting ofprimordial and primary follicles by VCD appears to be folliclestage-specific because no direct effects have been observed or measuredin larger (secondary to antral) follicles, or other non-ovarian tissuesas determined by necropsy, histopathology, plasma lipid profiles, andliver enzyme activity. Using a combination of molecular and cellularapproaches in our studies in rats we have collected evidence thatVCD-induced follicle loss is by acceleration of the normal rate ofatresia. These studies have also demonstrated that alterations inapoptosis-associated intracellular pathways activated by VCD dosing arespecific for small preantral follicles, as compared with large preantralfollicles or liver. Atresia in the rodent ovary occurs via apoptosis, orprogrammed cell death, and is a normal process without necrosis-inducedresponses such as inflammation. Evidence of VCD-induced impendingfollicle loss was observed as an increase in numbers of unhealthyfollicles in the treated group. The unhealthy appearance in VCD-treatedovaries is morphologically and ultrastructurally similar to unhealthyfollicles undergoing natural atresia in controls (14-16). At themolecular level, VCD-accelerated atresia in small preantral follicleswas identified because intracellular events associated with apoptosiswere measured selectively in the targeted follicular population. Theseevents include: A) increased Bax/BclxL ratio (17), B) increasedexpression of Bad, C) leakage of cytochrome c from mitochondria into thecytosol (18), D) increased caspase-3 activity (18), E) activation of theINK and p38 branch of the MAPK signaling pathway (19), F) retardation ofnatural atresia and bax expression (relative to controls) in smallfollicles following a single dose of VCD (20) and G) estrogenreceptor-mediated protection from VCD-induced follicle loss (21).Collectively, these data support at a molecular level that VCD causesfollicle loss by enhancing events associated with the normal process ofatresia. These molecular events are specific for the small preantralfollicles known to be physiologically targeted by VCD.

Follicle loss resulting from repeated dosing of F344 rats with VCD hasbeen well characterized (11-21). The conclusion from characterizationperformed in preliminary studies is that VCD causes the selective lossof primordial and primary follicles by accelerating the natural processof atresia via apoptosis. In long term studies in B6C3F₁ mice (22) andF344 rats (15) that were dosed with the parent compound,4-vinylcyclohexene (VCH, mice) or the ovotoxic form (VCD, rats),premature ovarian failure occurred within a year. However, the time ittakes from dosing of the animals until premature ovarian failure occursis too long in order to use animals prepared in such a manner as a modelfor menopause.

The development of an animal model that mimics the onset of menopause iscritical to enhance an understanding of the role of menopause in manyage-related diseases. By using VCD to chemically accelerate the normalprocess of atresia selectively in primordial and primary follicles, itwould be possible to more accurately approximate the physiologicalevents that occur during the progression from ovarian function throughimpending ovarian failure (perimenopause), to the eventual disease risksthat result after menopause. Therefore, such a model would be powerfulin scope because a wide variety of physiological and molecular endpointscan be designed for understanding the complexities of health risks thataccompany menopause in women.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a non-human femaleanimal suitable as a model of human perimenopause and/or menopause.

It is another object of the invention to provide methods of making theanimal model.

It is another object of the invention to provide a method of screeningan agent for its effect on a non-human female animal suitable as a modelof human perimenopause and/or menopause. In one specific aspect of thisembodiment, such a method provides for screening of agents which may beuseful for preventing or treating a condition associated with or causedby perimenopause and/or menopause.

It is another object of the present invention to provide methods ofinducing ovarian failure in mammalian female animals.

The objects of the present invention, and others, may be accomplishedwith a mammalian non-human female animal having at least a partialdepletion of ovarian primordial follicles and at least onecharacteristic of perimenopause and/or menopause induced byadministration of at least one compound selected from the groupconsisting of 4-vinylcyclohexene diepoxide (VCD), 4-vinylcyclohexene(VCH), 4-vinylcyclohexene-1,2-epoxide (VCH-1,2-epoxide), and4-vinylcyclohexene-7,8-epoxide (VCH-7,8-epoxide).

The objects of the present invention may also be accomplished with amethod of making the animal model described above by administering tothe animal an effective amount at least one compound selected from thegroup consisting of 4-vinylcyclohexene diepoxide, 4-vinylcyclohexene,4-vinylcyclohexene-1,2-epoxide, and 4-vinylcyclohexene-7,8-epoxide tocause at least a partial depletion of ovarian primordial follicles andat least one characteristic of perimenopause or menopause.

The objects of the present invention may also be accomplished with amethod of screening an agent, comprising:

administering an agent to the animal described above; and

evaluating the effect of the agent on the animal.

The objects of the present invention may also be accomplished with amethod of inducing ovarian failure in a non-human mammalian femaleanimal other than a mouse or a rat, comprising administering to theanimal an effective amount of at least one compound selected from thegroup consisting of 4-vinylcyclohexene diepoxide, 4-vinylcyclohexene,4-vinylcyclohexene-1,2-epoxide, and 4-vinylcyclohexene-7,8-epoxide.

The objects of the present invention may also be accomplished with amethod of sterilizing a mammalian non-human female animal other than amouse or a rat, comprising administering to the animal an effectiveamount of at least one compound selected from the group consisting of4-vinylcyclohexene diepoxide, 4-vinylcyclohexene,4-vinylcyclohexene-1,2-epoxide, and 4-vinylcyclohexene-7,8-epoxide.

The objects of the present invention may also be accomplished with amethod of controlling the size of a non-human mammalian animalpopulation, comprising administering an effective amount of at least onecompound selected from the group consisting of 4-vinylcyclohexenediepoxide, 4-vinylcyclohexene, 4-vinylcyclohexene-1,2-epoxide, and4-vinylcyclohexene-7,8-epoxide to the animal population sufficient tocause at least partial ovarian failure in at least a portion of thefemale members of the animal population.

The objects of the present invention may also be accomplished with asolid composition suitable for oral administration, comprising at leastone compound selected from the group consisting of 4-vinylcyclohexenediepoxide, 4-vinylcyclohexene, 4-vinylcyclohexene-1,2-epoxide, and4-vinylcyclohexene-7,8-epoxide and a solid excipient.

The objects of the invention may also be accomplished with a compositionsuitable for dermal or subcutaneous delivery to an animal, comprising atleast one compound selected from the group consisting of4-vinylcyclohexene diepoxide, 4-vinylcyclohexene,4-vinylcyclohexene-1,2-epoxide, and 4-vinylcyclohexene-7,8-epoxide in adermal or subcutaneous delivery device.

A more complete appreciation of the invention and many of the attendantadvantages thereof will be readily obtained as the same becomes betterunderstood by reference to the following figures and detaileddescription.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Effect of treatment with VCD on numbers of primordial andprimary follicles in mice. Female B6C3F1 mice (28 days of age) wereinjected (i.p.) with sesame oil control or VCD 80 mg/kg (1, 2, or 3times, ×, daily), 160 mg/kg (1 or 2× daily), or 240 mg/kg (1× daily) for15d. Ovaries were collected on d15 following onset of treatment andprocessed for histological evaluation as described in the Examplesbelow. Values represent mean number of follicles in the total of every20^(th) section±SEM; n=6 per group, different letters within a follicletype, different from one another (p<0.05).

FIG. 2: Effect of VCD-induced follicle loss over time. Female B6C3F₁mice (28 days of age) were injected daily with VCD (160 mg/kg, i.p. 1×)or sesame oil control for ≦15d. Ovaries were collected (d8-d46)following onset of treatment and processed for histological evaluation.Oocyte-containing follicles were classified and counted as described inthe Examples below. A) d8-30, primordial and primary follicles; B) d46primordial, primary, secondary and antral follicle numbers. Valuesrepresent the mean total number of follicles counted in every 20^(th)section of each ovary±SEM; n=4 per group, *p<0.05 or different letterswithin a follicle type, different from one another (p<0.05).

FIG. 3: Effect of VCD treatment on Caspase 3 activity in primordial andprimary follicles. Female B6C3F₁ mice (28 days of age) were injecteddaily with VCD (80 or 160 mg/kg, i.p.) or sesame oil for 10d. Ovarieswere collected 4 hours following the final treatment of VCD. Smallpre-antral follicles were isolated, and caspase-3 activity determined asdescribed in the Examples below. Data are represented as group meanvalues, n=2 replicates per group of 6 ovaries, different letters withina treatment concentration, different from one another (p<0.05.

FIG. 4: Effect of VCD treatment on circulating levels of FSH and antralfollicles. Female B6C3F1 mice (age 28 days) were injected daily withsesame oil or VCD (160 mg/kg, i.p., 1×15d). A) Plasma was collected ond15, d37, d46, d58, d100 and d120 following onset of treatment for 15dfor determination of FSH content as described in the Examples below.(n-6-18 per group, *p<0.01 different from control different letterswithin a treatment, different from one another p<0.05). B) Ovaries werecollected on d15, d30, d37, and d46 and processed for histologicalevaluation as described in the Examples below. Antral follicles werecounted in every 20^(th) section and a regression analysis was performedto compare circulating levels of FSH vs Antral follicle number (n=6-12per group, r²=0.87, p<0.02).

FIG. 5: Effect of VCD treatment on circulating levels of 17β-estradiol.Female B6C3F1 mice (age 28 days) were injected daily with sesame oilvehicle or VCD (160 mg/kg, i.p., 1×, 15d). Plasma was collected andpooled from a minimum of 12 animals per groups on d37, d46, d58 and d91following the onset of treatment and concentration of 17β-estradiol wasdetermined as described in the Examples below. (n=3 pools, *p<0.01different from control).

FIG. 6: Relative amounts of circulating 17β-estradiol andandrostenedione on d46 following the onset of treatment. Female B6C3F1mice (age 28 days) were injected daily with sesame oil vehicle or VCD(160 mg/kg, i.p., 1×, 15d). Plasma was collected and pooled from aminimum of 12 animals per group on d46 and concentration of17β-estradiol and androstenedione was determined as described in theExamples below. (n=3 pools).

FIG. 7: Effect of VCD treatment on circulating levels of Osteocalcin andbone histomorphometry. Female B6C3F1 mice (age 28 days) were treateddaily with sesame oil or VCD (160 mg/kg, i.p., 15d). Plasma wascollected on d46, d58, and d120 and concentration of osteocalcin wasmeasured as described in the Examples below. Data are represented as agroup mean values±SEM (n=6-10 per group, *p<0.05). Photomicrographs ofrepresentative femurs collected on d58 from B) VCD-treated and C)control animals, prepared for histological evaluation as described inthe Examples below. Arrows indicate distance from growth plate to distalmetaphysis of femur (n=6; Magnification 40×).

FIG. 8: Comparison of changes in reproductive parameters betweenVCD-dosed mice and women as they approach menopause. Summarized patternsof cyclycity and hormone levels measured in VCD-treated mice arecompared with those values reported in women. Values for mice areassigned day (d) relative to the onset of treatment, (d1) with VCD for15d.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based, in part, on the discovery that VCDinduces ovarian failure by acceleration of atresia in female animals andproduces, at most, only minimal effects on other body organs andphysiological pathways. In addition, if sufficient time is allowed topass, these animals develop one or more characteristics of perimenopauseor menopause. Thus, as discussed below, other than a reduction inovarian and uterine weight, there are no other direct irreversibleeffects observed at the tissue level. Subsequent physiologicalconditions that arise will be the consequence of ovarian failure.

As a result, these animals can be used as a model for humanperimenopause and menopause. Such a model system is dramaticallysuperior to animal models based on ovariectomy, irradiation, or geneticmanipulation. In addition, the procedures described herein can be usedto provide a method for non-surgical induction of ovarian failure tocontrol the populations of domestic and wildlife animals. A key featureof the animals of the present invention is that they contain theirovaries, i.e., the animals have not been subjected to ovariectomy.Therefore, the animals of the present invention contain at least onefunctional ovary, and preferably all of them.

As used herein the term “perimenopause” refers to a condition thatprecedes menopause in which ovarian cyclicity becomes irregular, FSHlevels become elevated, and ovarian 17β-estradiol levels become erratic.In women this can be up to 10 years before menstruation ceases. The term“menopause” as used herein refers to the time at which complete ovarianfailure has occurred. This is when menstrual periods have ceased, LH andFSH levels are elevated and 17β-estradiol levels have plummeted. Thus,the characteristics of perimenopause and menopause include thosedescribed above.

For perimenopause, histological evaluation of ovarian sections reveals acomplete absence of primordial follicles, and reductions in largerpreantral and antral follicles. At that time, circulating FSH levelsbecome elevated, and ovarian cyclicity has become irregular asdetermined by vaginal cytology. In addition, there is a loss of bonemineral density and a reduction in ovarian weight that begins inperimenopause and can continue during menopause. For menopause, allovarian follicles are depleted, circulating FSH levels have plateaued,and the animals are acyclic.

The model animal of the present invention is a mammalian non-humanfemale. Suitable animals include laboratory and research primates androdents. Specific examples of suitable animals include dogs, cats,hamsters, rabbits, sheep, cattle, deer, elk, moose, pigs, goats,ferrets, horses, monkeys, chimps, rats, mice. Rats and mice areespecially preferred.

Transgenic, gene-deficient (i.e., knock-out), or knock-in animals,particularly mice, are especially preferred for the study of certainconditions. The purpose of using these animals is that they model humandisease states which are associated relevant to the onset ofperimenopause and/or menopause. Preferred examples are mice and ratswhich have been modified in a property which is relevant to the changesin the body upon the onset of menopause and/or during menopause.Specific examples of such animals include those with modifications inapo e, apob, LDLR, LCAT, A^(Y), Foxnl^(nu), Bmp4, Kit, ERKO, BERKO,OB/OB, and Aβ.

The present invention also provides methods of preparing the animalmodel. VCD appears to be the active agent in producing the depletion ofovarian primordial follicles, or at least serves a precursor to theactual active species in vivo. Therefore, VCD may be administered to theanimals. Since VCH is converted to VCD in vivo, VCH may also be used inthe present invention. The pathway from VCH to VCD appears to involvethe formation of VCH-1,2-epoxide and/or VCH-7,8-epoxide. See Fontaine etal., Drug Metabolism and Disposition, 29, 179-184, 2001, FIG. 1 of whichis incorporated herein by reference. Therefore, VCH-1,2-epoxide and/orVCH-7,8-epoxide may also be administered in order to produce the animalof the present invention. In principle, any agent that is converted toVCD (or the actual active agent produced therefrom) in vivo may be usedto prepare the animal model.

As described by Fontaine et al. (see especially FIG. 1), VCH is a chiralcompound and each enantiomer is converted into epoxide products havingdiffering stereochemistry. All of these stereoisomers may be used in thepresent invention. Thus, the compound used may be (S)—VCH, (R)—VCH,(S)—VCH-1,2-expoxide, (R)—VCH-1,2-expoxide, (S)—VCH-7,8-expoxide,(R)—VCH-7,8-expoxide, (S)—VCH-diepoxide, (R)—VCH-diepoxide, and anymixture thereof. VCD, VCH, and VCH-1,2-epoxide are available fromAldrich Chemical Co.

The dosing of the compound, in terms of both dosage amount and method ofadministration, may vary widely. The compound is administered at asufficient dosage to cause at least partial depletion of the ovarianprimordial follicles. In so doing, a sufficient amount of time ispermitted to elapse so that the animal develops at least onecharacteristic of perimenopause or menopause. Thus, the production ofthe animal model depends on the method of delivery, the dosage amount,and the time that the dose is administered. For example, a continuousdose administered via an implant (discussed in more detail below) mayuse less VCD over the same time period as compared to repetitive i.p.injections.

In general, VCD is more potent than VCH and therefore may be used at alower dose. The potency of VCH-1,2-expoxide and VCH-7,8-expoxide isexpected to be between VCH and VCD.

In one embodiment, VCD is administered at a dose of at least 80 mg/kgper day. One preferred range is 80-720 mg/kg per day. This rangeincludes all specific values and subranges therebetween, such as 90,100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, and 700mg/kg per day. One preferred subrange is 100 to 160 mg/kg/day. Thisrange includes all specific values and subranges therebetween, such asat least 110, 120, 130, 140, 150, and 160 mg/kg/day. The preferred timeperiod for administering VCD is at least 10 days, preferably 10 to 30days, or more. In a preferred embodiment, VCD is administeredintraperitoneally (i.p.). In another embodiment, VCD is administeredusing an implant with a slow time release over several days.

A suitable dosage of VCH is at least 900 mg/kg/day. Preferably themaximum dose is at most 1600 mg/kg/day. This range includes all specificvalues and subranges therebetween, such as 1100, 1200, 1300, 1400, and1500 mg/kg/day. The preferred time period for administering VCH is atleast 6 days, preferably 10 to 30 days, or more. In a preferredembodiment, VCH is administered intraperitoneally (i.p.). In anotherembodiment, VCD is administered using an implant with a slow timerelease over several days.

Administration of the compound may be through any of the followingroutes: intraperitoneal injection, subcutaneous injection, lavage, oralintake, dermal patch, dermal application, dermal injection, inhalation,intravenous injection, transplacental exposure, intravaginal implant,subcutaneous implant, nasal spray, or implant dart. The exact dosage forany particular method of administration can be determined readily usingtechniques well-known in the art. A preferred example of a subcutaneousimplant is a minipump, particularly an Alzet minipump. The implant maybe biodegradable or non-biodegradable. Preferably, for use a populationcontrol device, the implant is biodegradable. For animals used aresearch models, the implant is preferably non-biodegradable.

A variety of different vehicles may be used to administer the compound.Examples of suitable vehicles include physiologically acceptable oils(e.g., sesame oil, corn oil, and mineral oil), DMSO, acetone, andmixtures thereof

Some routes of administration may be more effective in a commercialsetting as compared to other methods. For example, using a subcutaneousimplant may be more cost-effective in terms of animal handling andstorage and personnel costs associated with producing the animal incommercial quantities.

The animal model of the present invention may be used in a wide varietyof assays of screening agents for their potential effect on aperimenopausal or menopausal female. In this embodiment, the agent isadministered to the animal and the effect on the animal is evaluated.For example, the model can be used to evaluate, i.e., screen, potentialtherapeutic agents for preventing or treating conditions associated withperimenopause and menopause. In this embodiment, the agent isadministered to the animal and evaluated for its effect.

These types of screens are routine in the art. What the presentinvention provides is a novel model with which to perform the testing.For an example of a procedure for using an animal model of humanperimenopause and/or menopause to screen agents, see the ovariectomizedmouse model for human menopause described in U.S. Pat. No. 6,583,334,the contents of which are incorporated herein by reference.

Transgenic, gene-deficient, or knock-in animals are particularlypreferred in some aspects of this embodiment. Here, the effect of agenetic modification can be studied in isolation, i.e., no externalagents are administered to the animal, or the effects of a potentialtherapeutic agent can be evaluated as described above. Specific examplesof conditions include hot flashes, osteoporosis, incontinence,poylcystic ovarian disease, Alzheimer's disease, depression, maculardegeneration, arthritis, anxiety, obesity, ovarian cancer, diabetesmellitus, vaginal dryness, vaginal discharge, cancers of thereproductive tract, breast cancer, thinning of the skin, loss of libido,colorectal cancer, alopecia, hirsutism, cardiovascular disorders (whichinclude heart attack, stroke, deep vein thrombosis, hypertension,hypotension, ischemia, pulmonary embolism, atherosclerosis, heartabnormality, hypercholesterolemia, hypertriglyceridemia,hypocholesterolemia, hypotriglyceridemia, vascular defects, vascularhomeostasis, and sudden cardiac death), loss of manual dexterity,osteopenia, cognitive impairments, dementia, etc.

The present invention also provides a method of inducing ovarian failurein a mammalian non-human female animal other than a mouse or a rat,comprising administering to the animal an effective amount of thecompound. Suitable animals include those discussed above suitable dosagefor this purpose is at least 90 mg/kg per day of VCD for a timesufficient to induce ovarian failure. VCH can also be used for thispurpose, for example, at the dosages described above.

The compound may also be used to control the size of a non-human animalpopulation, comprising administering an effective amount of the compoundto the animal population sufficient to cause at least partial ovarianfailure in at least a portion of the female members of the animalpopulation. Such animals include dogs, cats, hamsters, ferrets, rabbits,sheep, cattle, horses, pigs, deer, elk, moose, bears, goats, monkeys,wild felines.

In this embodiment, administration of the compound may be through any ofthe following routes: intraperitoneal injection, subcutaneous injection,lavage, oral intake, dermal patch, dermal application, dermal injection,inhalation, intravenous injection, transplacental exposure, intravaginalimplant, subcutaneous implant, nasal spray, or subcutaneous dart.

The present invention also provides a solid composition suitable fororal administration, comprising the compound and a solid excipient.Prefereably, such a composition is in the form of a pill, capsule, orcaplet that can be administered to animals and humans. Solid excipientswhich are suitable for orally administerable compositions arewell-known. For a description, see Kirk-Othmer Encyclopedia of ChemicalTechnology, Fourth Edition, Volume 18, p. 491-502 (1996), incorporatedherein by reference.

The composition may also be in the form of a dermal or subcutaneousimplant. Such a composition contains the compound in a suitable dermalor subcutaneous deliver device. Such devices are well-known in the art,see Kirk-Othmer Encyclopedia of Chemical Technology, Fourth Edition,Volume 8, p. 445-474 (1996), incorporated herein by reference. Inanother embodiment, an implant dart could be used for wildlifepopulation control.

Because of the potential benefits of utilization of transgenic andgene-deficient mice developed to investigate specific pathologiesassociated with perimenopause and/or menopause, mice were chosen for theinitial development and characterization of the model of VCD-inducedovarian failure. In developing a model for menopause, it is critical toinduce follicle loss in a timely manner to provide sufficient time forlong-term studies. Preliminary experiments in immature d28 B6C3F₁ micewere conducted using an increased dosing regimen to more rapidlyaccelerate the rate of follicle loss (FIG. 1). A time course wasconducted to determine the minimum amount of dosing conditions necessaryto fully deplete the ovary of primordial follicles in B6C3F₁ micewithout producing adverse physiological side effects (FIG. 2). Animalswere treated daily with VCD via injection at 160 mg/kp/day i.p. orsesame oil for 15 days. Animals were killed, tissues harvested, andserum collected on d1-15, d37, d46, d58, d120, and d127 following theonset of dosing. To verify that follicle loss in this dosing regimen isalso via atresia, the activity of caspase 3, an enzyme involved in theapoptotic pathway, was measured in small preantral follicles isolated asdescribed in Hu et al. (18) following 10d of dosing (FIG. 3). Caspase-3activity was increased (p<0.05) in small pre-antral (primordial andprimary) follicles isolated from VCD-treated (14.37 flourescent units/40μg protein±4.9) relative to control (1.87 flourescent units/40 μgprotein ±0.53) mice indicating that the number of follicles undergoingatresia was increased. Thus, the increased dosing regimen with VCDrelative to 80 mg/kg/day (the dose previously used to characterizeVCD-induced follicle loss) did not impair intracellular apoptoticsignaling as would result if a toxic, necrotic response had occurred.The morphological appearance of primordial and primary follicles inVCD-treated animals on d10 was also indicative of atresia via apoptosis.From this experiment, the optimal dose of VCD was determined for 15d andsubsequent experiments were conducted using that dosing regimen. Todetermine the impact of loss of primordial follicles on ovarianfunction, antral follicles were counted and circulating FSH levelsmeasured on d15-d127 (FIG. 4). There was an inverse correlation (p<0.02)between these two parameters. This is indicative of impending ovarianfailure resulting from loss of large antral follicles. Throughout theexperiment several physiological parameters were measured. Body andtissue weights were evaluated. The onset of vaginal opening (31±0.5days), whole body weights and adrenal, spleen and kidney wet-tissueweights were not affected by VCD treatment at any time-point. The modestincreases in (p<0.05) liver weights seen in VCD-treated animals on dayd15 (10% above control) and d37(15% above control), however by d46,liver weights had returned to control levels. The modest increases(p<0.05) in liver weights seen in VCD-treated animals during the periodof exposure was because of activation of metabolic mechanisms.Circulating liver enzymes aspartate aminotransferase (AST) and plasmaalanine aminotransferase (ALT) in VCD-treated animals were not differentfrom controls at any time d1-d46 (p>0.05). Hepatocellular vacuolardegeneration (a pathologic lesion consistent with mild hepatocellulartoxicity) was evaluated by the University Animal Care DiagnosticLaboratory of the Arizona Health Sciences Center. There was nodifference in the occurrence of these lesions in naive, vehicle-treatedcontrol, or VCD-treated mice at any time. Ovarian tissue weights werenot significantly different across treatment groups on d10, or d15,however, by d37, VCD-dosed animals displayed significantly reducedovarian weights compared to controls. Uterine weights were also reducedby VCD treatment by d15. The reduced uterine weight likely obviouslyresulted from lower ovarian 17β-estradiol, and, therefore, the loss ofits lack of well-known tropic effect on the uterus. The decrease inovarian and uterine weights is consistent with that seen at 360d in thelong term studies in mice and rats and is indicative of ovarian failure;loss of large antral follicles and ovarian and uterine atrophy (15, 22).Collectively, these observations demonstrate that after 15d of VCDdosing, ovarian and uterine weight reductions are the only directirreversible physiological effects observed at the tissue level. Both ofthese findings are consistent with ovarian failure in menopausal women.Furthermore, no toxicity or impact on liver function was measured inblood or observed morphologically. It can therefore be assumed that thedevelopment of any subsequent adverse physiological conditions is theresult of ovarian failure.

It is to be noted that in any embodiment of the invention describedabove, 4-vinylcyclohexene diepoxide, 4-vinylcyclohexene,4-vinylcyclohexene-1,2-epoxide, and 4-vinylcyclohexene-7,8-epoxide asdescribed above can be used alone as the agent for inducing ovarianfailure. Alternatively, these compounds can be used together in anydesired ratio.

Examples

Having generally described this invention, a further understanding canbe obtained by reference to certain specific examples which are providedherein for purposes of illustration only and are not intended to belimiting unless otherwise specified.

Example 1

Treatment with VCD reduced (p<0.05) the number of primordial and primaryfollicles 15d following commencement of treatment in a dose-dependentmanner in B6C3F₁ mice (FIG. 1). Primordial follicles were reduced at 80mg/kg to 12.1% of control, 160 mg/kg (80 mg/kg, 2× daily, and 160 mg/kg1× daily) 0% of control, 240 mg/kg (80 mg/kg 3× daily) 2.1% of control,240 mg/kg (240 mg/kg 1× daily) 0% of control, and 320 mg/kg (160 mg/kg2× daily) 0% of control. Primary follicles were reduced at 80 mg/kg to38.6% of control, 160 mg/kg (160 1× daily) 2.8% of control, 160 mg/kg(80 mg/kg 2× daily) 11.3% of control, 240 mg/kg (80 mg/kg 3× daily) 5.1%of control, 240 mg/kg (240 mg/kg 1× daily) 0% of control and 320 mg/kg(160 mg/kg 2× daily) 0.6% of control.

Treatment with VCD (160 mg/kg, i.p.) for=15 days reduced (p<0.05)primary and primordial follicle numbers progressively over time (FIG.2A). By d46 following commencement of 15d of treatment, all folliclepools were substantially depleted (p<0.05) relative to control. Therewere no primordial follicles and primary, secondary and antral follicleswere 0.5%, 0.7%, and 2.6% of control values respectively (FIG. 2B).

During the initiation of apoptosis, the executioner caspases (caspase-3)become activated which results in cellular collapse. Mice were givendaily injections of VCD (160 mg/kg, or 80 mg/kg, i.p.) for 10d. Fourhours after the final treatment, small preantral follicles were isolatedand activity of the apoptosis-associated executioner protease enzymecaspase-3 was measured. Relative to controls, increased (p<0.05)cleavage activity of caspase-3 in isolated small pre-antral follicleswas observed in a dose-associated manner (FIG. 3).

The optimal concentration of VCD for primordial follicle loss (lowestamount, shortest time) was determined to be 160 mg/kg, injected 1× daily(resulting in complete loss of primordial follicles). Daily treatmentfor 15d with VCD (160 mg/kg) subsequently resulted in a progressivereduction (p<0.05) of ovarian weights on d37 (71.2% of control), d46(61.8% of control) and d120 (20.7% of control) following the onset oftreatment (Table 1). Additionally, uterine weight relative toage-matched vehicle controls was reduced (p<0.05) on d15 (66.04%), d37(71.8%), and d120 (47.0%). There was no effect of VCD treatment onadrenal, kidney or spleen weight at any time. A modest increase (p<0.05)in liver weight was observed on d15 (10% above control) and d37 (15%above control). These weights had returned to control levels by d46.Because VCD can be metabolized by the liver, liver function wasinvestigated by measurement of the liver enzymes aspartateaminotransferase (AST) and alanine aminotransferase (ALT) andhepatocellular vacuolar degeneration was evaluated (Table 2). Plasmasamples were collected on d1-d46 (following 15d of treatment) andcirculating levels of ALT and AST were determined to be within thenormal range for this strain of mice. Further, to assess hepatic effectson circulating lipid profiles, total plasma cholesterol, HDL andtriglycerides were determined for d10, d15 and d37 following the onsetof treatment (Table 2). All lipid fractions were within the normal rangefor this species, and did not differ (p<0.05) from control.

VCD treatment (160 mg/kg, 15d) resulted in increased circulating levelsof FSH compared to controls over time (FIG. 4A). On d37 following theonset of treatment, plasma FSH levels were increased (p<0.05) inVCD-dosed animals (11.5±5.7 ng/ml) relative to control (1.7±0.06 ng/ml),with a continued increase on d46 (VCD: 14.3±1.3 ng/ml), d58 (VCD:20.2±1.2 ng/ml), d100 (VCD: 25.8±0.23 ng/ml), and d120 (VCD: 25.9±1.1ng/ml). Plasma FSH levels on d127 (data not shown) was increased(p<0.05) relative to controls, however, not increased relative to d100and d120 values indicating a plateau in circulating FSH.

Antral follicle numbers were progressively reduced (d15-d46; p<0.05) asa result of VCD treatment (160 mg/kg, 15d) and negatively correlated(r²=0.87) with increasing circulating levels of FSH on those days (FIG.4B). Because antral follicles had become depleted, d58 and subsequenttime points are not included in the analysis.

Plasma levels of 17β-estradiol in VCD-treated animals were below thelimit of detection in VCD-treated groups on d37 and remained at thatlevel through d91 (FIG. 5). Conversely, in control animals circulating17β-estradiol began to increase (p<0.05) on d46 (2.05 pg/ml), d57 (2.21pg/ml) and d91 (2.21 pg/ml). Circulating levels of androstenedione inplasma pools from VCD-treated animals were similar to controls on d46(control 0.64±0.01; VCD: 0.73±0.05 ng/ml). Because of this, there was anincreased relative abundance of androgen versus estrogen (FIG. 6;control: 24%; VCD: 97% of androgen plus estrogen).

VCD treatment (160 mg/kg, i.p.) resulted in disruption of estrouscyclicity. The mean cycle length was determined in control animals to be4.49±0.48d. Animals with cycle lengths less than 3d or greater than 5dwere determined to be irregular. Cycle length was disrupted in VCD-dosedanimals beginning on d15 (7/24, irregular) and by d58 all animals wereacyclic. Control animals consistently maintained regular cycles acrossall time points.

Osteocalcin is a protein produced in osteoblasts and is increased incirculation under conditions of increased bone turnover. Plasma levelsof osteocalcin were increased (p<0.05) in VCD-treated (160 mg/kg, i.p.,15d) groups relative to control on d46 (control: 109.6±7.0, VCD:131.3±5.4 ng/ml), and d58 (control: 111.2±6.9, VCD: 156.3±6.1 ng/ml FIG.7A). By d120 the two groups were not different (control: 127.1±12.7,VCD; 143.4±7.1 ng/ml; FIG. 7A). Femora collected on d58, fromVCD-treated animals (160 mg/kg, 15d, FIG. 7B) displayed enhanceddistances from growth plate to distal metaphysis of the femur withincreased lucane relative to controls (FIG. 7C).

FIG. 8 is a comparison of the patterns of cyclicity, circulating levelsof FSH, osteocalcin, and 17β-estradiol between VCD-treated mice(d1-d100) and values published for similar stages of reproductivefunction in women. For example, in women, puberty is equivalent to d3following the onset of treatment in mice, and peri-menopause can becompared to d10-d50, with post-menopause resembling d50-d100.

Materials and Methods

Animals: Immature female B6C3F₁ mice (21 days) were obtained from HarlanLaboratories (Indianapolis, Ind.), housed in plastic cages, andmaintained on 12 hour light-dark cycles at 22±2° C. Animals were allowedto acclimate to the animal facilities for 1 week prior to initiation oftreatment. Food and water were available ad libitum. All experimentswere approved by the University of Arizona Institutional Animal Care andUse Committee and conformed to the Guide for the Care and Use ofExperimental Animals.

Treatment: Immature mice (d28) were weighed and injected i.p. witheither 80 mg/kg (1, 2, or 3 times daily), 160 mg/kg (1 or 2 timesdaily), or 240 mg/kg (once daily) with VCD or sesame oil (vehiclecontrol, i.p.) for 15 d resulting in a total daily exposure of 80, 160,240, or 320 mg/kg (n=6/group). All reagents were obtained fromSigma-Aldrich (St. Louis, Mo.). Estrous cycles of each animal weremonitored daily by vaginal cytology from date of vaginal opening topersistent diestrus in VCD-treated mice, and from date of vaginalopening to d120 in controls. On d8, d10, d12, d14, d15, d30, d37, d46,d57 and d120, animals were weighed, euthanized by CO₂ inhalation, andthe ovaries, liver and trunk blood were collected. Plasma was separatedfrom whole blood and stored at −20° C., and liver tissues were stored at−80° C.

At each time point, adrenals, kidneys, uteri, ovaries, liver, and spleenwere removed, grossly examined for lesions and wet tissue weights wererecorded. Additional blood samples were collected by retro-orbitalpuncture and plasma was collected and stored on d58, d79, d91, and d100.

Histology and oocyte counting: Ovaries were trimmed of fat and placed inBouin's fixative (2 hours), transferred to 70% ethanol,paraffin-embedded and serially sectioned (4-5 μm), mounted and stainedwith hematoxylin and eosin. In every 20^(th) section (10-13 sections perovary), pre-antral follicles were classified as primordial (oocytesurrounded by a single layer of flattened granulosa cells), primary(oocyte surrounded by a single layer of cuboidal cells), secondary(oocyte surrounded by multiple layers of granulosa cells) or antralfollicles (follicles containing a fluid-filled antrum). Femora werecollected on d58 from both control and VCD-treated animals, fixed in 4%paraformaldehyde, transferred to formaldehyde/formic acid (Cal-Rite,Richard Allan Scientific, Kalamazoo, Mich.), decalcified 24 hours,vacuum embedded in paraffin and 4.5 μm sections of distal metaphysiswere prepared and stained with hematoxylin and eosin.

Follicle Isolation: Small preantral follicles (fraction 1, 25-100 pm indiameter), were prepared by gentle enzymatic dissociation of ovaries andhand sorting with micropipettes. Pools of follicles were prepared fromboth ovaries of six mice in each treatment (control or VCD) for eachobservation. Following isolation, follicles were washed twice with M199medium and stored at −20° C.

Hormone Assays: Plasma FSH was measured by radioimmunoassay (RIA). RatFSH hormone antigen, rat FSH antiserum and mouse FSH referencepreparation were purchased from the National Institute of Diabetes andDigestive and Kidney diseases. Iodination reagents (Iodo-Beads™28665,28666) were purchased from Pierce (Rockford Ill.). Briefly, a standardcurve was prepared and cold standards and samples (100 μl) were added tolabeled tubes along with primary antibody (1:1400 dilution) andiodinated FSH. Samples were shaken and stored at 4 C overnight. On day2, 100 μl secondary antibody (Sigma Chemical, cat. #R9133) was added(1:10) dilution along with 200 μl of 2% normal rabbit serum andincubated at room temperature for 5 minutes. Tubes were centrifuged for15 minutes at 3000 rpm, supernatant was decanted and pellets werecounted in a gamma counter for 1 minute each. All samples were run induplicate. The mean sensitivity of the assay was 200 pg/ml and inter-and intra-assay coefficients of variation were 2.7% and 6.7%respectively. Plasma 17β-estradiol was measured in unextracted plasma byradioimmunoassay (Diagnostic Products Inc., Calif.). The meansensitivity of the assay was 5.1 pg/ml. The intra-assay coefficient ofvariation was 5.3%. Circulating androstenedione was measured by specificRIA. Androstenedione standards and antibody were provided by Dr. CherylDyer (Northern Arizona University). The mean sensitivity of the assaywas 70 pg/ml with an inter-assay coefficient of variation of 5.3%. Theresults of all RIAs were calculated by four-parameter logistic analysisusing the software AssayZap (BioSoft, Ferguson, Mo.). Results are themean±SEM.

Assessment of Hepatic Function: Hepatocellular vacuolar degeneration andenzymatic activity of circulating aspartate aminotransferase (AST) andalanine aminotransferase (ALT) were determined by the DiagnosticLaboratory of the Arizona Health Sciences Center. Total cholesterol andtriglycerides were quantified using Sigma reagents 401-25P and 344-20respectively. HDL was measured by precipitating apoB-containinglipoproteins (Boehringer Mannheim reagent 543004) and assayingcholesterol in the supernatant fraction using the Sigma 401-25P reagent.

Caspase-3 Protease Activity Measurement: The cleavage activity ofcaspase-3 was measured by a fluorescent activity assay. Briefly, theenzymatic reaction was carried out at 37° C. in protease assay buffer(20 mM HEPES, 100 mM NaCl, 10 mM dithiothreitol, 1 mM EDTA, 0.1% [w/v]3-3 ([3-cholamidopropyl]dimethylammonio)-1-propanesulfanate, and 10%sucrose pH 7.2). Cellular protein (60-180 μg) was incubated with 50 μMof caspase-3 substrate, DEVD-AMC, at 37° C. for 60 minutes. Substratecleavage was detected by measurement of the fluorescence of free7-amino-4-methylcoumarin (AMC) with an F-2000 fluorescencespectrophotometer (Hitachi, Ltd., Tokyo, Japan) at 460 nm emission uponexcitation at 380 nm.

Osteocalcin measurement: Intact serum Osteocalcin was measured using animmunoradiometric assay (IRMA, Immutopics, San Clemente, Calif.). Assaysensitivity was 0.4 ng/ml and the intra-assay coefficient of variationwas 6.7%.

Data Analysis: Oocyte numbers were determined in ovaries from individualanimals, averaged, and the means (±SEM) in control versus treatedanimals were analyzed for significant differences by one-way analysis ofvariance (ANOVA) with significance set at p<0.05. Post-hoc tests(Tukey-Kramer) were used where appropriate. The differences in plasmahormone concentrations among treatments were analyzed using a one-wayANOVA. The correlation coefficient was determined by regressionanalysis.

Example 2 Range Finding Experiment with Alzet Minipumps to Determine theNo-Observable-Adverse-Effect-Level (NOAEL) of VCD and Verify Primordialand Primary Follicle Depletion

We will use B6C3F₁ mice because they are the most completelycharacterized mouse strain using multiple injections of VCD. The 160mg/kg/d dose injected i.p. for 15d yields the greatest acceleration offollicle depletion via a single injection without adverse effects. Weknow that if the total VCD dose delivered over 15d (2400 mg/kg) isinjected in a single bolus the mice die within hours. Mice can toleratemore VCD per day if it is metered out over the 24 h time period. Ourpreliminary data indicates that mice tolerate VCD well when dosed 3times a day @80 mg/kg/d (total 240 mg/kg/d). Three injections a day, permouse, is labor intense and may be impractical for commercialproduction.

VCD is a member of the 4-vinylcyclohexene family of chemicals andappears to be the bioactive compound in rats and mice. It is rapidlymetabolized into the inactive tetrol and polar products. Thus far, onlyrepeated injection of VCD has been examined for its promotion offollicular depletion. Our goal is to reduce the 15 daily injections toone procedure or injection per mouse. To reach this goal we must firstdetermine whether continuous delivery of VCD will elicit folliculardepletion similar to the repeated dosing regimen.

To examine the effect of continuous VCD delivery, Alzet minipumps model1002 will be implanted subcutaneously (sc.). Although repeated injectionof VCD has always been performed via an intraperitoneal route, for easeand less stress to the animal we will implant the minipumps sc.Subcutaneous administration of VCD will be an effective route as we knowdermal application of VCD in acetone is active and i.p. injection of VCDin DMSO vehicle achieves the same follicular depletion as i.p. injectionof VCD in sesame oil. It is even possible that sc. administration mayincrease the efficacy of VCD as it will not be rapidly inactivated byits first pass metabolism through the portal system which most likelyoccurs when injected i.p.

The effective plasma concentration of VCD has not been determined. VCDdoes not absorb visible or UV light and is too small (140.2 molecularweight) to be tagged with a large molecule such as biotin to follow itsdisposition. Because VCD is not easily detected, the peak concentrationof VCD in blood after its injection has not been determined. Intravenousadministration of ¹⁴C-tagged VCD shows its rapid conversion into theinactive tetrol form. VCD's half life is 4.4 minutes and its meanresidence time is 4.7 minutes. For our purposes we do not need toachieve a target plasma concentration because our goal is operational,to cause follicle depletion without causing generalized toxicity.

Given the short half life of VCD in blood it may be that continuousdelivery via minipump will require less VCD to deplete ovarianfollicles. Significant reduction in effective concentration when goingfrom injection to infusion is frequently observed. For instance,continuous administration of endostatin by i.p. minipumps decreased theeffective dose by 8-10 fold as well as increasing its efficacy. Itcannot be assumed that continuous delivery of VCD will increase itsefficacy because this must be specifically determined for each chemical.VCD is inexpensive and readily available from numerous suppliers sominimization of VCD use is not a consideration. Instead, the goal is toreduce the number of injections per mouse to reduce stress to theanimals, personnel related expenses and the per diem cost for mice incommercial facilities.

Alzet minipump model 1002 will be used to continuously administer VCD.Osmotic minipumps deliver their contents at a constant μl/hr rate.Therefore, to adjust the dose we will change the amount of VCD loadedinto the minipump. The first dose tested will be the closestapproximation to the dose administered in the 15 daily injections of 160mg/kg/d (2400 mg/kg). Using Alzet minipump model 1002 which delivers0.25 μl/hr, it will take approximately 14 days to deliver 95% of the 100μl reservoir volume.

For the first pilot experiment there will be 6 cycling female mice (aged40) in each of 4 groups. The groups will be; vehicle control receiving15 daily injections of 50% DMSO, positive control receiving 15 dailyinjections of VCD in 50% DMSO @160 mg/kg/d, vehicle minipump infusionwith 50% DMSO, and minipump infusion with VCD in 50% DMSO. The VCDconcentration in the minipump will be adjusted for the weight of eachmouse. For instance for a 25 gm mouse the delivery rate will be 0.188mg/hr for 14 days which will deliver continuously the same total amountof VCD delivered by 15 daily injections received by the positive controlgroup. The minipump reservoir is compatible with 50% DMSO. Although VCDis water soluble, diluting it in DMSO will be a more efficient vehicleto work with when filling the minipumps.

Alzet 1002 minipumps will be placed sc. in mice under anesthesia usingprocedures described by the manufacturer. Mice will be monitored for 24h after implantation to watch for normal feeding and behavior. Vehicleand VCD filled minipumps will be implanted the same day as injectiongroups receive their first dose. All mice will be weighed daily andobserved for signs of general toxicity such as wasting, reduced activityand reduced grooming. On d15 after the onset of dosing all mice will bekilled by exsanguination under anesthesia. Blood will be collected bycardiac puncture, and plasma separated and stored at −20° C. Ovarieswill be collected, trimmed free of fat and fixed in Bouin's solution.Uteri will be collected and weighed to use as a bioassay for whole bodylevels of estradiol. Adrenals, kidneys, and liver will be removed,weighed, and analyzed for gross stress effects. Liver samples will becollected and prepared for histopathologic examination.

If the continuous delivery of VCD at an equivalent dose of 160 mg/kg/dproduces adverse effects, then the experiment will be repeated usinghalf the VCD dose (80 mg/kg/d) which will be achieved by using Alzetminipump model 1002 with half as much VCD concentration loaded into thereservoir. If the 80 mg/kg/d dose also results in adverse effects, wewill reduce the dose again by half. This will be done until a dose isfound that establishes the NOAEL.

Plasma will be analyzed for the liver enzymes alanine aminotransferase(ALT) and aspartate aminotransferase (AST). If liver function isaffected by VCD continuous administration then these activities will besignificantly elevated in the VCD minipump group versus the vehicleinjection and minipump administration. Increased FSH is measured byradioimmunoassays (RIA: sensitivity=200 pg/ml), and is a good index ofreduced 17-beta estradiol production by the ovary. It is unclear that15d of VCD exposure will reduce the number of estradiol producingfollicles so we will determine if FSH levels are significantly elevated.Follicle counts on sectioned and stained ovaries will be done. Daily VCDinjections (160 mg/kg/d) for 15d will result in a 100% reduction inprimordial follicle populations and we anticipate that the same willoccur in the VCD minipump group. We expect the DMSO injection andminipump control groups will have the same number of primordialfollicles. The goal of this experiment is to determine the NOAEL forcontinuous VCD administration. This dose will be used in Experiment 3 tocompare the relative efficacy of VCD given in repeated injection versuscontinuously.

Example 3 Using the NOAEL from Example 2 to Define the Kinetics ofFollicle Depletion and Increase in Plasma FSH, and Determining if LessVCD is Required When Administered by Continuous Delivery

Completion of the Example 2 will provide evidence that continuousdelivery of VCD by minipump will not cause general toxicity andaccomplish follicle depletion similar to 15 daily VCD injections. TheVCD NOAEL will be used to analyze the detailed time course of follicledepletion and increased plasma FSH.

The VCD NOAEL will be used in Alzet minipump model 1002 that deliversthe reservoir volume in 14 days. There will be 4 groups of d40 femalemice, 6 mice/group, killed at each time point. Groups 1 and 2 willreceive 15 daily injections of 50% DMSO vehicle and 50% DMSO with VCD(160 mg/kg, i.p.). Groups 3 and 4 will receive minipumps implanted sc.loaded with 50% DMSO vehicle and 50% DMSO with VCD. Mice will bemonitored for 24 h after the implantation to watch for normal feedingand behavior. On the day of minipump implant, the injection controlgroups will receive the first VCD dose. All mice will be weighed dailyand watched for signs of general toxicity such as wasting, reducedactivity and reduced grooming. The rate of follicle depletion and FSHincrease will be determined by killing mice on day 3, 6, 9, 12, 15 and30 after the onset of injection and infusion dosing. The mice that willbe kept until day 30 will have their minipump implants removed underanesthesia on day 21 as suggested by the manufacturer. Minipumps must beremoved because once dosing is complete, irritating concentrated saltsolution can leak into surrounding tissue. Animals will be killed andtissues harvested and analyzed as described in Example 2.

As before, plasma ALT and AST activities will be measured to gauge livertoxicity. In this experiment with a 30 day time point we expect thatplasma FSH will increase significantly and perhaps sooner in VCD infusedmice. Primordial and primary follicle populations will be depleted byday 30 in the VCD treatment groups but the relative rate may differbetween injection and infusion groups. A 100% follicle depletion ofprimordial and primary follicles is a desirable end-point as it predictstotal follicle depletion with 45 days. We may observe that continuousVCD delivery accelerates follicle depletion compared to repeated VCDinjection. If this is observed we will adjust the NOAEL to a lowerconcentration as the lowest dose of VCD is desirable.

Example 4 Using the VCD NOAEL from Example 3 and Determining if TimeFrame of its Administration can be Less Than 14d

Our goal is to reduce mouse per diem cost by shortening the time frameof VCD dosing. We will use the effective VCD dose and administer it inAlzet minipumps that deliver 95% of their reservoir volume in 1 day, 3days, 7 days and 14 days. Mice can tolerate more VCD when administeredin more frequent lower doses (preliminary data FIG. 4). Thus weanticipate that shortening the dosing time frame will be tolerated bythe mice making the generation of animal model more economical andfeasible for commercial purposes.

There will be 2 groups of d40 female mice, 6 mice/group, that havevehicle or VCD minipumps implanted. In this experiment only minipumpswill be used, no injections will be used to administer vehicle/VCD. TheVCD NOAEL will be used to fill Alzet minipump models as described below,and deliver 50% DMSO vehicle or VCD in 50% DMSO in 1 day model 2001D=8.0μl/hr, 3 days model 1003D=1.0 μl/hr, 7 days model 1007D=0.5 μl/hr, and14 days model 1002=0.25 μl/hr. Mice will be monitored for 24 h after thesc. implantation to watch for normal feeding and behavior. All mice willbe weighed daily and watched for signs of general toxicity such aswasting, reduced activity and reduced grooming. All mice will besacrificed on day 15. Blood will be collected by cardiac puncture, andplasma separated and stored at −20° C. Ovaries will be collected,trimmed free of fat and fixed in Bouin's solution. Tissues will beharvested and analyzed as previously described. Liver samples will becollected and prepared for histopathologic examination.

As before, plasma ALT and AST activities will be measured to gauge livertoxicity. Accelerated VCD delivery may make mice sick and we willcarefully monitor them for signs of poor tolerance and performeuthanasia where necessary. Since mice injected 3 times a day with 80mg/kg/injection tolerated VCD as well as mice injected once a day with160 mg/kg we expect that the minipump time frame shortened to 7 dayswill be tolerated by the mice. It is difficult to predict the outcomesfor mice receiving the full VCD dose continuously in 1 or 3 days. Miceinjected with the full VCD dosed in one bolus died so perhaps infusionin 24 hours will also kill the mice.

Example 5 Capturing the Peri and Post Menopause Endocrine Windows Using½ and 2/4 of the Dose Identified in Experiment 3 with the Time FrameDetermined from Example 4

To this point we have used VCD dosing that accelerates follicledepletion to achieve complete ovarian failure or partial folliculardepletion without causing general toxicity.

Reducing VCD dose slows the rate of follicle depletion so that a perimenopause like state can be generated. To our knowledge there is noother mouse model that can simulate the endocrine status ofperi-menopause. Our animal model provides the opportunity to studydiseases that begin during peri-menopause such as loss of bone mineraldensity seen in osteoporosis. We anticipate users who want toinvestigate their diseases in peri-menopausal model mice. To providethis model variation we will define the pharmacokinetics of VCDcontinuous delivery that protracts the rate of follicle depletionleading to a peri-menopause like endocrine state.

There will be 4 groups of d40 female mice, 6 mice/group, that havevehicle or VCD loaded minipumps implanted. The VCD dosing time framewill define the Alzet minipump model to be used. Group 1 will beimplanted with 50% DMSO vehicle, group 2 will receive the dose, group 3will receive ½ the dose of group 2 and group 4 will receive ¼ the doseof group 2. Mice will be monitored for 24 h after the sc. implantationto watch for normal feeding and behavior. All mice will be weighed dailyand watched for signs of general toxicity such as wasting, reducedactivity and reduced grooming. Mice will be sacrificed on days 15, 30and 60. Blood will be collected by cardiac puncture, and plasmaseparated and stored at −20° C. Tissues will be harvested and analyzedas previously described.

Since we are reducing the VCD NOAEL in this experiment we will not needto monitor liver toxicity so will not measure ALT or AST or performhistopahtologic examination. We will count follicles in ovarian sectionsand measure FSH in the plasma samples for each time point and VCD dosetested. We anticipate that less VCD will reduce the rate of follicledepletion which will be reflected in higher follicle counts at each timepoint and FSH will not rise as quickly as observed with the highest doseof VCD. We will compare the rates of follicle depletion for each VCDdose by graphing the % of follicles left with each dose over time toderive the slope of the line that provides the depletion rate.

Accomplishment of this work will demonstrate feasibility of transitionfrom a 15d i.p. injection protocol to a single-procedure infusionprotocol, that results in a peri- and post-menopausal mouse model.Minipumps are too expensive, at >$20/minipump, and require surgicalprocedures to implant/explant and therefore, may not be feasible forcommercial production of the animal model of the present invention.Development of a cost-effective protocol will facilitatecommercialization of the model.

General Methods for Examples 2-5

Animals: Female B6C3F₁ mice will be obtained from JAX (Sacramento,Calif.), housed in plastic cages, and maintained on 12 hour light-darkcycles at 22±2° C. Animals will acclimate to the facilities for 1 weekprior to treatment. Food and water will be available ad libitum.

Treatment: Mice will be weighed to determine VCD dose for minipumpsand/or injection with VCD (15 d, sc, 160 mg/kg, Sigma-Aldrich, St.Louis, Mo.) or 50% DMSO (vehicle control, n=6/group). Whole blood willbe collected by cardiac puncture and plasma separated and stored at −20°C. On kill dates, animals will be weighed, euthanized and the ovaries,uteri, adrenals, kidneys, spleen, and livers collected and weighed.

Histology and Follicle Counting: Ovaries will be trimmed free of fat andplaced in Bouin's fixative (2 hours), transferred to 70% ethanol,paraffin-embedded and serially sectioned (4-5 pm), mounted and stainedwith hematoxylin and eosin. In every 20^(th) section, follicles will beclassified as previously described.

Assessment of Hepatic Function: Hepatocellular histopathology andenzymatic activity of circulating AST and ALT will be determined by theDiagnostic Laboratory at the Arizona Health Sciences Center.

Hormone Assay: Plasma FSH will be measured by RIA according toinstructions from the National Hormone and Pituitary DistributionProgram. Samples will be assayed in duplicate. Sensitivity of the assayis 200 pg/ml. Results will be calculated by four-parameter logisticanalysis using the software AssayZap (BioSoft, Ferguson, Mo.).

Data Analysis: The effect of VCD treatment on follicle number will bedetermined by student's t test between two group means for parametricdata, and for nonparametric data by Kruskal-Wallis. Tissue weights,liver enzyme levels, and plasma hormone concentrations, will be averagedfor each treatment and the means (±SEM) in control versus treatedanimals will be analyzed for significant differences by one-way analysisof variance (ANOVA). Post-hoc tests (Tukey-Kramer) will be used whereappropriate. Tests for homogeneity of variance (Bartlett's) andnormality (Shapiro-Wilk) will be routinely performed to assure theassumptions of the ANOVA are met. To determine theno-observed-adverse-effect level (NOAEL), effects of treatment on totalbody weights, and follicle counts will be analyzed by Student's t-test.The incidence scores of the histopathological data will be analyzed bythe Fisher exact test. Significance for all tests will be set at p<0.05.

Vertebrate Animals:

1) Female B6C3F₁ will be used because the preliminary data has beencollected in this strain. All experimental protocols with animals willbe conducted at Northern Arizona University under IACUC approvedprotocols. Litters of 10 female B6C3F₁ pups at 21 days of age with anursing mother will be obtained (Jackson Laboratories, Bar Harbor, Me.).The number of animals used is estimated to be the minimum required toperform these experiments without wasting animals because of inadequatesampling size. NOTE: each animal can serve as a separate n; however, inview of the fact that most animals may be maintained for an extendedperiod of time, animal numbers have been increased by 20% to allow foranimal loss. Experiments will be performed on an ongoing basis, and mostanimals will be maintained for an average of 60 days. Numbers of animalsused are indicated in the methods and design for each experiment. Oncereceived, animals will be allowed to acclimate one week prior to theonset of dosing. Animals will be kept in plastic cages (4/cage),maintained on 12-h light/12-h dark cycles (22° C.) and provided food(Purina rat chow) and water ad libidum. On d 40 of age, animals will bedosed daily for 15 days with and sc. injection of vehicle control 50%DMSO, or 4-vinylcyclohexene diepoxide (VCD) or implanted with vehicle orVCD loaded Alzet osmotic minipumps per the manufacturers instructions.At the appropriate time, animals will be killed by pentobarbitaloverdose (245 mg/kg, i.p.), and blood drawn and tissues removed forhistological evaluation.

2) The laboratory mouse is well-defined as a research animal.Preliminary studies to determine the optimal dosing conditions forinducing ovarian failure have been conducted in the B6C3F₁ strain.Therefore, the proposed studies will continue with this strain. Thenumber of animals requested has been carefully calculated to providesufficient amounts of blood and tissue samples for the experimentsplanned. The numbers calculated have also taken into account appropriatemeasurements for statistical strength. The experiments proposed do notduplicate previous studies performed by us, or reported by others.

3) All animal care will be provided under the supervision of aboard-certified laboratory veterinarian of NAU's PHS approved laboratoryanimal facility. All procedures will be approved by the Northern ArizonaUniversity Institutional Animal Care and Use Committee.

4) All procedures with animals are designed to cause minimal distress ordiscomfort. Animals will receive daily sc. injections of vehicle (50%DMSO) or test compound (VCD) for 15 days or less. This route ofinjection has been chosen because previous work has demonstratedreproducible follicle loss is produced. However, follicle loss alsooccurs with VCD following oral, dermal, or inhalation exposure. Overall,the sc. route of exposure provides minimal trauma to the test animals,requires no sophisticated equipment (such as exposure chambers) andreduces risk of exposure of personnel. Subcutaneous implantation ofAlzet osmotic minipumps will be performed under pentobarbital anesthesiaand the wound will be closed with clips or sutures. The mice will beclosely observed for signs of distress or infection. All personnelinvolved have been trained to humanely accomplish these tasks.

5) All animals will be euthanized by pentobarbital overdose (245 mg/kg,i.p.). This method is consistent with the recommendations of the Panelon Euthanasia of the American Veterinary Association.

REFERENCES

-   1. Lobo R, Kelsey J, Marcus R. Menopause: biology and pathobiology.    1 ed. San Diego: Academic Press; 2000.-   2. Mosca L, Manson J E, Sutherland S E, Langer R D, Manolio T,    Barrett-Connor E. Cardiovascular disease in women: statement for    healthcare professionals from the American Heart Association.    Circulation 1997; 96:2468-82.-   3. Oparil S. Hormones and Vasoprotection. Hypertension 1999;    33(II):170-6.-   4. Hulley S, Grady D, Bush T. Randomized trial of estrogen plus    progestin for secondary prevention of coronary heart disease in    post-menopausal women. Heart and Estrogen/progestin Replacement    Study (HERS) Research Group. JAMA 1998; 280:605-13.-   5. Writing Group for the Women's Health Initiative Investigators.    Risks and benefits of estrogen plus progestin in healthy    postmenopausal women. J. Amer. Med. Assoc. 2002; 288:321-333.-   6. Purdie D W. Consequences of long-term hormone replacement    therapy. Brit. Med. Bull. 2000; 56:809.-   7. Rodriguez C, Pantel A V, Calle E E, Jacob E J, Thun M J. Estrogen    replacement therapy and ovarian cancer mortality in a large    prospective study of US women. J. Amer. Med. Assoc. 2001; 285:1460.-   8. Erickson G F. Ovarian anatomy and physiology. In: Lobo R, Kelsey    J, Marcus R, editors. Menopause, Biology and Pathobiology. 1 ed. San    Diego: Academic Press; 2000. p 13-31.-   9. Bellino F L. Nonprimate animal models of menopause: Workshop    report. Menopause 2000; 7(1):14-24.-   10. Judd H L. Hormonal dynamics associated with the menopause.    Clinical Obstetricts and Gynecology 1976; 19(4):775-88.-   11. Flaws J A, Doerr J K, Sipes I G, Hoyer P B. Destruction of    preantral follicles in adult rats by 4-vinyl-1-cyclohexene    diepoxide. Reproductive Toxicology 1994; 8(6):509-14.-   12. Kao S-W, Sipes I G, Hoyer P B. Early effects of obotoxicity    induced by 4-vinylcyclohexene diepoxide in rats and mice.    Reproductive Toxicology 1999; 13(1):67-75.-   13. Smith B J, Mattison D. R., Sipes I G. The role of expoxidastion    in 4-vinylcyclohexene-induced ovarian toxicity. Toxicology and    Applied Pharmacology 1990; 105:372-81.-   14. Springer L N, McAsey M E, Flaws J A, Tilly J L, Sipes I. G.,    Hoyer P B. Involvement of apoptosis in 4-vinylcyclohexene diepoxide    induced ovotoxicity in rats. Toxicol Appl Pharmacol 1996    August;139(2):394-401.-   15. Mayer L P, Pearsall N A, Christian P J, Devine P J, Payne C M,    McCuskey M K, Marion S L, Sipes I. G., Hoyer P B. Long-term effects    of ovarian follicular depletion in rats by 4-vinylcyclohexene    diepoxide. Reproductive Toxicology 2002; In press.-   16. Devine P J, Payne C M, McCuskey M K, Hoyer P B. Ultrastructural    evaluation of ooxyted during atresia in rat ovarian follicles. Biol.    Reprod. 2000; 63:1245-1252.-   17. Hu X, Christian P J, Sipes I. G., Hoyer P B. Expression and    redistribution of cellular Bad, Bax, and Bcl-X(L) protein is    associated with VCD-induced ovotoxicity in rats. Biology of    Reproduction 2001 November;65(5):1489-95.-   18. Hu X, Christian P J, Thompson K E, Sipes I G, Hoyer P B.    Apoptosis induced in rats by 4-vinylcyclohexene diepoxide is    associated with activation of the caspase cascades. Biology of    Reproduction 2001; 65(1):87-93.-   19. Hu X, Flaws J A, Sipes I. G., Hoyer P B. Activation of    Mitogen-Activated Protein Kinases and AP-1 Transcription Factor in    Ovotoxicity Induced by 4-vinylcyclohexene Diepoxide in Rats. Biology    of Reproduction 2002; In press.-   20. Borman S M, VanDePol B J, Kao A, Thompson K E, Sipes I G, Hoyer    P B. A single dose of the ovotoxicant 4-vinylcyclohexene diepoxide    is protective in rat primary ovarian follicles. Toxicology and    Applied Pharmacology 1999; 158:244-52.-   21. Thompson K E, Sipes I. G., Greenstein B D, Hoyer P B.    17-estradiol affords protection against 4-vinylcyclohexene    diepoxide-induced ovarian follicle loss in Fisher-344 rats.    Endocrinology 2002; 143(3):1058-65.-   22. Hooser S B, Douds D P, DeMerell D G, Hoyer P B, Sipes I G.    Long-term ovarian and gonadotropin changes in mice exposed to    4-vinylcyclohexene. Reproductive Toxicology 1994; 8(4):315-23.

Obviously, numerous modifications and variations of the presentinvention are possible in light of the above teachings. It is thereforeto be understood that within the scope of the appended claims, theinvention may be practiced otherwise than as specifically describedherein.

1. A mammalian non-human female animal having at least a partialdepletion of ovarian primordial follicles and at least onecharacteristic of perimenopause and/or menopause induced byadministration of 4-vinylcyclohexene diepoxide at a dosage of at least100 mg/kg/day or 4-vinylcyclohexene at a dosage of at least 1000mg/kg/day.
 2. The animal of claim 1, which is prepared by a processcomprising administering to the animal 4-vinylcyclohexene diepoxide at adosage of at least 100 mg/kg/day.
 3. The animal of claim 1, which isprepared by a process comprising administering to the animal4-vinylcyclohexene at a dosage of at least 1000 mg/kg/day.
 4. The animalof claim 1, which is suitable as a model of perimenopause.
 5. The animalof claim 1, which is suitable as a model of menopause.
 6. The animal ofclaim 1, wherein the at least a partial depletion of ovarian primordialfollicles and at least one characteristic of perimenopause and/ormenopause is induced by said 4-vinylcyclohexene diepoxide.
 7. The animalof claim 1, wherein the at least a partial depletion of ovarianprimordial follicles and at least one characteristic of perimenopauseand/or menopause is induced by said 4-vinylcyclohexene.
 8. The animal ofclaim 1, wherein the animal has loss of bone mineral density.
 9. Theanimal of claim 1, which has at least one characteristic ofperimenopause.
 10. The animal of claim 1, which has at least onecharacteristic of menopause.
 11. The animal of claim 1, wherein said atleast one characteristic of menopause is irregular ovarian cyclicity,elevated FSH levels, erratic ovarian 17β-estradiol levels, loss of bonemineral density, or reduced ovarian weight.
 12. The animal of claim 1,wherein said at least one characteristic of menopause is depletion ofovarian follicles, menstrual periods have ceased, elevated LH levels,elevated FSH levels, diminished ovarian 17β-estradiol levels, loss ofbone mineral density, or reduced ovarian weight.
 13. The animal of claim1, which is a mouse.
 14. The mouse of claim 13, which is transgenic. 15.The mouse of claim 13, which is gene-deficient.
 16. The mouse of claim13, which is a knock-in.
 17. The animal of claim 1, which is transgenic.18. The animal of claim 1, which is gene-deficient.
 19. The animal ofclaim 1, which is a knock-in.
 20. The animal of claim 1, which is a rat.21. The animal of claim 1, which is a primate.
 22. The animal of claim1, which is a canine.
 23. A method of preparing the animal of claim 1,comprising administering to the animal 4-vinylcyclohexene diepoxide at adosage of at least 100 mg/kg/day or 4-vinylcyclohexene at a dosage of atleast 1000 mg/kg/day.
 24. The method of claim 23, wherein said4-vinylcyclohexene diepoxide is administered to the animal.
 25. Themethod of claim 24, wherein the 4-vinylcyclohexene diepoxide isadministered intraperitoneally (i.p.), subcutaneously (s.c.), or by animplantable device. animal.
 26. A method of claim 23, wherein said4-vinylcyclohexene is administered to the
 27. The method of claim 26,wherein the 4-vinylcyclohexene is administered intraperitoneally (i.p.),subcutaneously (s.c.), or by an implantable device.
 28. The method ofclaim 23, wherein the animal is suitable as a model of perimenopause.29. The method of claim 23, wherein the animal is suitable as a model ofmenopause.
 30. The method of claim 23, wherein the animal is a mouse.31. The method of claim 30, wherein the mouse is transgenic.
 32. Themethod of claim 30, wherein the mouse is gene-deficient.
 33. A method ofscreening an agent, comprising: administering an agent to the animal ofclaim 1; and evaluating the effect of the agent on the animal.
 34. Themethod of claim 33, wherein the agent is a treatment for one or moreconditions selected from the group consisting of hot flashes,osteoporosis, incontinence, poylcystic ovarian disease, Alzheimer'sdisease, depression, macular degeneration, arthritis, anxiety, obesity,ovarian cancer, diabetes mellitus, vaginal dryness, vaginal discharge,cancers of the reproductive tract, breast cancer, thinning of the skin,loss of libido, colorectal cancer, alopecia, hirsutism, cardiovasculardisorders, loss of manual dexterity, osteopenia, cognitive impairments,and dementia.
 35. The method of claim 34, wherein said cardiovasculardisorders are selected from the group consisting of heart attack,stroke, deep vein thrombosis, hypertension, hypotension, ischemia,pulmonary embolism, atherosclerosis, heart abnormality,hypercholesterolemia, hypertriglyceridemia, hypocholesterolemia,hypotriglyceridemia, vascular defects, vascular homeostasis, and suddencardiac death.
 36. The method of claim 34, wherein the animal is amouse.
 37. A method of inducing ovarian failure in a mammalian non-humanfemale animal other than a mouse or a rat, comprising administering tothe animal an effective amount of at least one compound selected fromthe group consisting of 4-vinylcyclohexene diepoxide,4-vinylcyclohexene, 4-vinylcyclohexene-1,2-epoxide, and4-vinylcyclohexene-7,8-epoxide.
 38. The method of claim 37, wherein saidcompound is 4-vinylcyclohexene diepoxide.
 39. The method of claim 37,wherein said compound is 4-vinylcyclohexene.
 40. The method of claim 37,wherein the animal is a canine.
 41. A method of controlling the size ofa mammalian non-human animal population, comprising administering aneffective amount of at least one compound selected from the groupconsisting of 4-vinylcyclohexene diepoxide, 4-vinylcyclohexene,4-vinylcyclohexene-1,2-epoxide, and 4-vinylcyclohexene-7,8-epoxide tothe animal population sufficient to cause at least partial ovarianfailure in at least a portion of the female members of the animalpopulation.
 42. The method of claim 41, wherein the animal is selectedfrom the group consisting of dogs, cats, hamsters, ferrets, rabbits,sheep, cattle, horses, pigs, deer, elk, moose, bears, goats, monkeys,and wild felines.
 43. The method of claim 41, wherein said compound is4-vinylcyclohexene diepoxide.
 44. The method of claim 41, wherein saidcompound is 4-vinylcyclohexene.
 45. A method of sterilizing a mammaliannon-human female animal other than a mouse or a rat, comprisingadministering an effective amount of at least one compound selected fromthe group consisting of 4-vinylcyclohexene diepoxide,4-vinylcyclohexene, 4-vinylcyclohexene-1,2-epoxide, and4-vinylcyclohexene-7,8-epoxide to the animal.
 46. The method of claim45, wherein the animal is selected from the group consisting of dogs,cats, hamsters, ferrets, rabbits, sheep, cattle, horses, pigs, deer,elk, moose, bears, goats, monkeys, and wild felines.
 47. The method ofclaim 45, wherein said compound is 4-vinylcyclohexene diepoxide.
 48. Themethod of claim 45, wherein said compound is 4-vinylcyclohexene.
 49. Asolid composition suitable for oral administration, comprising at leastone compound selected from the group consisting of 4-vinylcyclohexenediepoxide, 4-vinylcyclohexene, 4-vinylcyclohexene-1,2-epoxide, and4-vinylcyclohexene-7,8-epoxide and a solid excipient.
 50. Thecomposition of claim 49, wherein said compound is 4-vinylcyclohexenediepoxide.
 51. The composition of claim 49, wherein said compound is4-vinylcyclohexene.
 52. A composition suitable for dermal delivery of atleast one compound selected from the group consisting of4-vinylcyclohexene diepoxide, 4-vinylcyclohexene,4-vinylcyclohexene-1,2-epoxide, and 4-vinylcyclohexene-7,8-epoxidecontained in a dermal delivery device.
 53. The composition of claim 52,wherein said compound is 4-vinylcyclohexene diepoxide.
 54. Thecomposition of claim 52, wherein said compound is 4-vinylcyclohexene.55. A composition suitable for subcutaneous delivery, comprising atleast one compound selected from the group consisting of4-vinylcyclohexene diepoxide, 4-vinylcyclohexene,4-vinylcyclohexene-1,2-epoxide, and 4-vinylcyclohexene-7,8-epoxidecontained in a subcutaneous delivery device.
 56. The composition ofclaim 55, wherein said compound is 4-vinylcyclohexene diepoxide.
 57. Themethod of claim 55, wherein said compound is 4-vinylcyclohexene.
 58. Amammalian non-human female animal having at least a partial depletion ofovarian primordial follicles and at least one characteristic ofperimenopause and/or menopause induced by administration of at least onecompound selected from the group consisting of4-vinylcyclohexene-1,2-epoxide and 4-vinylcyclohexene-7,8-epoxide.
 59. Amethod of preparing the animal of claim 58, comprising administering tothe animal an effective amount at least one compound selected from thegroup consisting of 4-vinylcyclohexene-1,2-epoxide and4-vinylcyclohexene-7,8-epoxide to cause at least a partial depletion ofovarian primordial follicles and at least on characteristic ofperimenopause and/or menopause.
 60. A method of screening an agent,comprising: administering an agent to the animal of claim 58; andevaluating the effect of the agent on the animal.